4.1. Plant Material and Growth Conditions

Two susceptible wheat genotypes (Gatcher and Janz) and three resistant to moderately resistant wheat genotypes (CPI133872, GS50a, and QT8343) were grown as a glasshouse experiment at the Leslie Research Facility (27°32′02″ S 151°56′09″ E), Toowoomba, Australia, in August to October. Treatments were replicated three times in a factorial randomized block design with five genotypes, two nematode treatments (inoculated and non-inoculated), and five harvest time points (2, 4, 6, 8, and 10 weeks post-nematode inoculation (PNI)).

A self-mulching black vertisolic soil was collected from Formartin, Australia (27°22′12″ S 151°25′53″ E), and pasteurized in an air: steam stream at 85 °C for 45 min [61]. The soil moisture content was determined by drying 100 g of steam-pasteurized soil at 105 °C for 48 h. Slow-release fertilizer Osmocote^® Plus Trace Elements (Scott Australia, Baulkham Hills, Australia) was used at the rate of 1 g per pot of 330 g soil (oven-dried equivalent). The pots were placed on glasshouse benches fitted with a continuous bottom watering system with water tension maintained at 2 cm by a capillary system using bidim^® matting (Geofabrics Australasia, Brisbane, Australia) [62]. The inoculated treatments were separated from the non-inoculated treatments by a 5-cm gap between the capillary mats to avoid cross-contamination. Under-bench heating was used to maintain soil temperature at 22 ± 4 °C, the optimum temperature for P. thornei reproduction [63]. Soil temperature was monitored throughout the experiment using iButtons (Thermochron^®, Baulkham Hills, Australia) placed in arbitrarily selected pots at 3 cm of depth in the soil.

Two seeds were sown on a base layer of soil (80% of the total soil volume). Nematodes were added in a 10-mL liquid suspension to inoculated treatments, at the rate of 3300 P. thornei (J2: 23.5%, J3: 30%, J4: 16.5% adult 30%) equivalent to 10 nematodes/g of oven-dried soil per pot (70 mm square, 150 mm height). The seed and nematodes were covered with the remaining 20% soil [62]. Ten days after sowing, plants were thinned to one seedling per pot with a scalpel blade, retaining roots inside the pots. The roots of each genotype were assessed for total phenol content and oxidative enzymes (PPO and POD) at 2, 4, 6, and 8 weeks PNI. A final time-point at 10 weeks was used to assess nematode reproduction in the roots and soil for each genotype.

Wheat seeds of five genotypes (Gatcher, Janz, GS50a, QT8343 and CPI133872), were grown without P. thornei inoculation in a controlled environment growth cabinet (Bioline, Percival Scientific, IA, USA) for 3 weeks. The seeds were surface sterilized according to Wu et al. with slight modification [64]. The seeds were covered in 70% ethanol for 5 min followed by diluted bleach solution (NaOCl) (2.5%) for 15 min. The seeds were then rinsed with sterile Milli–Q^® (Merck, Darmstadt, Germany) reverse osmosis water five times. The seeds were imbibed by soaking in sterile Milli–Q^® water for 12 h, then placed on autoclaved petri plates containing moistened sterile filter paper and allowed to germinate for 48 h. Two-day old surface-sterilized and pre-germinated seedlings were transferred aseptically into Schott bottles (DURAN^® GLS 80, DWK Life Sciences, Wertheim, Germany) containing 200 mL of 0.3% autoclaved tap water agar [64]. Four seedlings were placed on the agar per bottle. The lids were screwed on loosely and then wrapped with parafilm. The experiment was arranged in a completely randomized design with three replicate bottles for each genotype.

Light intensity inside the growth cabinet was maintained at 400 µ mole/m² with a photoperiod of 13 h light and 11 h dark. The intensity of the light was measured with a photosynthetically active radiation (PAR) light meter (Li-250A, Li-COR Bio-Sciences, Lincoln, NE, USA). The light wavelength was in the PAR range of 400 to 700 nm. The temperature was maintained at 22 ± 2 °C throughout the experiment.

Twenty-one wheat genotypes were selected to cover a range of resistance/susceptibility to P. thornei [65] (Table 2). The wheat genotypes were grown using the same glasshouse conditions as described for Experiment 1. Treatments were replicated three times in a split plot design with plus or minus P. thornei inoculation randomized to the main plots and wheat genotypes randomized in the sub-plots in a row : column design. The plants were grown in the glasshouse for 8 weeks from July to September.

Origin and resistance rating to Pratylenchus thornei for wheat genotypes.

R = resistant, MR = moderately resistant, MS = moderately susceptible, S = susceptible, VS = very susceptible.